With the development of genetic science and genetic engineering, DNA synthesis technology plays an increasingly important role in the life sciences. The de novo synthesis of DNA assemblies, including the synthesis of regulatory sequences, whole genes, artificial metabolic pathways or even complete artificial genomes, will bring great changes to human life science research. Since the first oligonucleotide chain synthesis by a human in 1961 (Nirenberg et al. (1961) Proc. Natl. Acad. Sci. USA 54: 1588), DNA synthesis and assembly technologies have made great progress. At present, the synthesis and assembly of large DNA fragments are performed by way of assembling a number of oligonucleotide fragments below 100 bp due to certain technical limitations (Gibson, et al. (2010) Science 329: 52; Gibson. (2009) Nucleic. Acids Res. 37: 6984; Li & Elledge (2007) Nat. Methods 4: 251; Bang & Church (2008) Nat. Methods 5: 37; Shao et al. (2009) Nucleic Acids Res. 37: e16), and the associated cost is roughly stabilized at about 2.2 yuan per base pair (bp). Thus, traditional DNA synthesis and assembly methods, due to cost limitations, are difficult to apply to genomic DNA synthesis.
In 2004, using oligonucleotide microarray chips, Tian Jingdong et al. successfully synthesized 292 different oligonucleotide fragments on DNA chips, which were then assembled into a 14.6-kb DNA fragment. The foregoing work makes it feasible to perform chip-based large-scale DNA long-fragment synthesis and assembly with high efficiency and low cost (Tian (2004) Nature 432: 1050). In 2010, Kosuri et al. utilized Agilent's commercial DNA microarray chips to achieve the synthesis of hundreds of genes (Kosuri et al. (2010) Nat. Biotech. 28: 1295) and provide a complete and established technical route (Eroshenko et al. (2012) Curr. Protoc. in Chem. Biol. 4: 1). Based on this technology, the GEN9 Company was founded in the United States in 2012 and became the first commercial company to offer on-chip DNA synthesis services around the world. The price of its DNA synthesis products is about USD 0.26 per bp, which is lower than the market price of traditional DNA synthesis.
On-chip DNA synthesis technology is currently facing two major challenges, which are high error rates of the oligonucleotide chains, and the impact from the oligonucleotide library's high complexity on the assembly. Hence, further improvement on the accuracy rate of on-chip DNA synthesis technology and reduction in the cost for assembly and screening will play a crucial role in commercial applications of the on-chip DNA synthesis technology. In the technical route of the existing on-chip DNA synthesis technology, measurement of the accuracy rate of synthesis of DNA fragments and screening for accurate fragments are typically accomplished by way of first-generation sequencing technology, the Sanger sequencing process. Due to the technical bottlenecks of the technology of on-chip DNA synthesis, assembly products tend to be more complex when compared with the original designed sequence, that is to say the assembled DNA molecule can have multiple variations with high complexity in its sequence (including single base variation, nucleotide insertion and deletion, etc.), wherein the probability of single base variation is about 0.1% to 1% depending on the differences of platform and design. As a result, in the case that the single base error rate is 0.5%, when assembling a DNA fragment of 750 bp, the probability of obtaining a completely accurate DNA fragment in one synthesis is only 2.33%, the probability of having no more than one base error is 11.11%, and the probability of having no more than two base errors is 27.63%. As a result, in order to find an accurate molecule whose sequence is completely consistent with the designed sequence from the complex DNA assembly products and ensure a success rate of 90%, at least 98 monoclones have to be selected for Sanger sequencing. In the foregoing case, if the cost for each Sanger reaction is 20 yuan, the total cost for the foregoing process would be about 1,960 yuan, which is equal to 2.61 yuan per bp and is significantly higher than that of the traditional synthesis method. Moreover, in order to find at least one clone with no more than one base error and meanwhile ensure a success rate of 90%, 20 monoclones have to be selected for Sanger sequencing verification. In this case, the cost for such verification is about 400 yuan, which is equal to 0.53 yuan per bp. Further, in order to find at least one clone with no more than two base errors and meanwhile ensure a success rate of 90%, 8 monoclones have to be selected for Sanger sequencing verification. In this case, the cost for such verification is about 160 yuan, which is equal to 0.21 yuan per bp. However, when the clones selected have more than 3 base errors, the associated cost for material and time in the process of error removal would be considerably high. Accordingly, the on-chip DNA synthesis technology will lose its cost advantages. In light of the foregoing, it can be seen that the traditional method for measuring the accuracy rate of DNA chip synthesis products based on Sanger sequencing has a relatively high cost, which is the main source of the cost associated with the on-chip DNA synthesis technology, as well as one of the major bottlenecks of this technology to further reduce the cost.